Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May 10;7(19):27874-88.
doi: 10.18632/oncotarget.8442.

REG4 promotes peritoneal metastasis of gastric cancer through GPR37

Hexiao Wang  1 , Lei Hu  1 , Mingde Zang  1 , Baogui Zhang  2 , Yantao Duan  1 , Zhiyuan Fan  1 , Jianfang Li  1 , Liping Su  1 , Min Yan  1 , Zhenggang Zhu  1 , Bingya Liu  1 , Qiumeng Yang  1
Affiliations

REG4 promotes peritoneal metastasis of gastric cancer through GPR37

Hexiao Wang et al. Oncotarget. .

Abstract

Being the major reason of recurrence and death after surgery, peritoneal metastasis of gastric cancer dooms the prognosis of advanced gastric cancer patients. Regenerating islet-derived family, member 4 (REG4) is believed to promote peritoneal metastasis, however, its mechanism is still a moot point at present. In the present study, we show that high expression of REG4 correlates with advanced stage and poor survival prognosis for gastric cancer patients. REG4 overexpression significantly enhances peritoneal metastasis by increasing adhesion ability. Moreover, SP1 is proved to be a transcription factor of REG4 and induce REG4 expression upon TGF-alpha stimulation. Also, G protein-coupled receptor 37 (GPR37) is identified to be in the same complex of REG4, which mediates REG4's signal transduction and promotes peritoneal metastasis of gastric cancer cell. Interestingly, we also discover a positive feedback loop triggered by REG4, amplifying itself through EGFR transactivation, consisting of GPR37, ADAM17, TGF-alpha, EGFR, SP1 and REG4. In conclusion, REG4 promotes peritoneal metastasis of gastric cancer through GPR37 and triggers a positive feedback loop.

Keywords: REG4; adhesion; gastric cancer; peritoneal metastasis; positive feedback.

PubMed Disclaimer

Conflict of interest statement

We have no potential conflicts of interest to declare.

Figures

Figure 1
Figure 1. Expression of REG4 in clinical gastric cancer tissues
A. Negative REG4 expression in non-tumor gastric mucosa. B. Negative REG4 expression in gastric cancer. C. Weak positive REG4 expression in gastric cancer. D. Strong positive REG4 expression in gastric cancer. E. Kaplan-Meier survival curve of patients positive and negative for REG4 expression.
Figure 2
Figure 2. REG4 promotes adhesion and peritoneal metastasis of gastric cancer cells
A, B. mRNA and protein level of REG4 were evaluated by qRT-PCR and Western Blot in 9 gastric cancer cell lines and 1 immortalized gastric epithelial cell line. C, D. Cells or percentage adherent to different ECM components or Matrigel coated plates after 30min incubation were quantified at OD 560nm. E, F. Representative images and number of cells adherent to murine peritoneum after 30min incubation. G. Ascites volume and disseminated tumor number collected in mice. Error bars correspond to mean ± SD of at least three independent experiments. *P < 0.05, **P<0.01.
Figure 3
Figure 3. GPR37 is in the same complex with REG4
A. Immunoprecipitation of the whole cell extracts from SGC-7901/REG4-Flag and SGC-7901/Vector-Flag with anti-Flag antibody. B. Mass spectra results of the differential protein band in (A). C. Immunoprecipitation of the whole cell extracts from SGC-7901/REG4-Flag with anti-Flag antibody, and from SGC-7901/GPR37-HA with anti-HA antibody. D. Confocal microscopy for SGC-7901 cells stained with anti-REG4, anti-GPR37 antibody and iFluor 594 anti-rabbit IgG, Alexa488 anti-goat IgG second antibody. The cells were treated with PBS (row 1) or recombinant REG4 (rows 2–4), and were untransfected (row 2), transfected with control-siRNA (row 3), or GPR37-siRNA (rows4). Cell nuclei were counterstained with DAPI.
Figure 4
Figure 4. Knocking down GPR37 abrogates the pro-peritoneal metastasis effect of REG4
A, B. Cells or percentage adherent to different ECM components or Matrigel coated plates were quantified at OD 560nm after 30min incubation. C, D. Representative images and number of cells adherent to murine peritoneum after 30min incubation. E. Ascites volume and disseminated tumor number collected in mice. Error bars correspond to mean ± SD of at least three independent experiments. *P < 0.05, **P<0.01.
Figure 5
Figure 5. SP1 is the transcription factor of REG4 upon TGF-alpha stimulation in SGC-7901
A. Binding sites of SP1 on REG4 promoter were predicted by different software. B. TGF-alpha(10nM) increases REG4 on both mRNA and protein level. C. Knocking down SP1 abrogates REG4 inducement upon TGF-alpha stimulation. D. Phosphorylation level of EGFR, ERK, SP1 were analyzed after TGF-alpha(10nM) stimulation. C, E. Activation of EGFR pathway and expression of REG4 were tested with or without EGFR inhibitor, MEK inhibitor and SP1 siRNA, respectively, after TGF-alpha(10nM) stimulation. F. A brief scheme of wild type, truncation and mutation REG4 promoter cloned in pGL3-basic. G. Luciferase activity of a reporter construct harboring different REG4 promoter with or without TGF-alpha(10nM) stimulation. H. SP1 occupancy (fold enrichment) on REG4 promoter areas with or without TGF-alpha(10 nM) stimulation. Error bars correspond tomean ± SD of at least three independent experiments. *P < 0.05, **P<0.01.
Figure 6
Figure 6. Knocking down SP1 abrogates the pro-peritoneal metastasis effect of REG4
A, B. Cells or percentage adherent to different ECM components or Matrigel coated plates were quantified at OD 560nm after 30min incubation. C, D. Representative images and number of cells adherent to murine peritoneum after 30min incubation. E. Ascites volume and disseminated tumor number collected in mice. Error bars correspond to mean ± SD of at least three independent experiments. *P < 0.05, **P<0.01.
Figure 7
Figure 7. REG4 triggers a positive feedback loop
A. mRNA and protein level after transient transfection of REG4 plasmid. B. TGF-alpha concentration in medium supernatant with or without GPR37 siRNA or ADAM17 siRNA after rhREG4 (10ng/ml) stimulation. C, D. Phosphorylation and total level of ADAM17 and EGFR with or without GPR37 siRNA or ADAM17 siRNA after rhREG4(10ng/ml) stimulation. E. A brief scheme of the positive feedback loop triggered by REG4.
See this image and copyright information in PMC

References

    1. Herrero R, Park JY, Forman D. The fight against gastric cancer - the IARC Working Group report. Best Pract Res Clin Gastroenterol. 2014;28:1107–1114. - PubMed
    1. Kagawa S, Shigeyasu K, Ishida M, Watanabe M, Tazawa H, Nagasaka T, Shirakawa Y, Fujiwara T. Molecular diagnosis and therapy for occult peritoneal metastasis in gastric cancer patients. World journal of gastroenterology : WJG. 2014;20:17796–17803. - PMC - PubMed
    1. Thomassen I, van Gestel YR, van Ramshorst B, Luyer MD, Bosscha K, Nienhuijs SW, Lemmens VE, de Hingh IH. Peritoneal carcinomatosis of gastric origin: a population-based study on incidence, survival and risk factors. Int J Cancer. 2014;134:622–628. - PubMed
    1. Hartupee JC, Zhang H, Bonaldo MF, Soares MB, Dieckgraefe BK. Isolation and characterization of a cDNA encoding a novel member of the human regenerating protein family: Reg IV. Biochim Biophys Acta. 2001;1518:287–293. - PubMed
    1. Li FY, Ren XB, Xu EP, Huang Q, Sheng HQ, Lv BJ, Lai MD. RegIV expression showing specificity to gastrointestinal tract and its potential role in diagnosing digestive tract neuroendocrine tumor. J Zhejiang Univ Sci B. 2010;11:258–266. - PMC - PubMed

MeSH terms